1. Field of the Invention
The invention relates to the use of .beta.-alethine and/or its corresponding monosulfide, .beta.-alethine, for the inducement of cell differentiation, adaptation of cells to culture, and enhancement of cell phenotypic expression, vitality, longevity, and production. In particular, the invention relates to the use of .beta.-alethine for inducing the differentiation of precursor cells into specialized cells, for normalizing function of malfunctioning cells, and for eliminating intractable cells (cells which are "resistant to cure, relief, or control", Dorland's Illustrated Medical Dictionary, 26th Edition, 1974, W. B. Saunders, Philadelphia, Pa., U.S.A.).
Cellular differentiation is a well-known phenomenon which broadly refers to processes by which precursor cells (commonly termed "stem cells") develop into specialized cells. Differentiation compounds, i.e., those factors which induce cell multiplication, in the literature (see, e.g., "Growth, Differentiation, and the Reversal of Malignancy", Scientific American, pp. 40-47, January, 1986, and the publications cited therein) and the implications of each with respect to therapeutic use in the treatment of disease or disorders of the body are of much current interest. The present application relates to the identification of .beta.-alethine as a non-cell-lineage-dependent differentiation compound, and the use of .beta.-alethine to induce differentiation and/or normalization of the function of a variety of cells, particularly for therapeutic benefits.
"Phenotypic cell expression" is defined herein as the manifestation of an entire range of physical, biochemical, and physiological characteristics of an individual cell as determined both genetically and environmentally, in contrast to "genotypic cell expression", which in the art solely refers to the expression of the cell chromosomal sequence. [See, for example, Dorland's Illustrated Medical Dictionary, 26th Edition, 1974, W. B. Saunders, Philadelphia.] Biological activity of the compounds of the invention thus includes modulation of the expression of genetic material of cells in culture as influenced by the condition and environment of each cell, including the age of the cell, the culture or conditions employed, and the presence of optionally added biological effectors.
2. Discussion of Related Art
.beta.-alethine is an endogenous thiol known to be produced in vivo as a byproduct of metabolic pathways. It is related via these pathways to pantothenic acid, which is a vitamin having known nutritional benefits (see, e.g., J. Reprod. Fert. 57: 505-510 [1979]), and related compounds have been suggested for use in conjunction with radiotherapy as radioprotectors (J. Med. Chem. 29: 2217-2225, 1986; WO 85/00157, Jan. 17, 1985). No other relevant asserted biological functions of this compound are known to be described in the prior art. The compound is primarily well-known as a starting material for the chemical synthesis of related compounds (see, e.g., Japanese patent applications (83) 198461; (83) 46063A2; (81) 156256A2; (81) 104861A2; (80) 124755; (75) 62932; (80) 07222; and U.S. Pat. Nos. 2,835,704 and 4,552,765; for examples of the preparation of .beta.-alethine, .beta.-alethine, pantetheine, and its derivatives or intermediates, and also for Coenzyme A and Coenzyme A derivatives or intermediates).
It is well-known that endogenous thiols and disulfides are critical to the function of a multitude of thiol- and disulfide-dependent branch-point enzymes controlling access to major metabolic pathways. Glutathione (GSH, gamma-glutamyl cysteinylglycine, an acid tripeptide thiol) is the most abundant thiol in mammalian cells, and an entire regulatory and regenerating system ensures an adequate supply of this reducing agent (3,4,5), which maintains and buffers cell thiol/disulfide ratios. Coenzyme A (CoA) and lipoic acid are prevalent in mammalian systems and also regulate dependent enzyme activity. Xenobiotic thiols such as dithiothreitol (DTT, Cleland's reagent) or dithioerythritol are routinely used experimentally to regulate activity of thiol-dependent enzymes.
In response to demand, thiols such as GSH, CoA, and lipoic acid can, for example, activate thiol-activatable enzyme by reducing inactive oxidized (disulfide) enzyme to the corresponding thiol with a concomitant oxidation of the activating thiol to its corresponding disulfide (GSSG in the case of glutathione-GSH) according to the following scheme, wherein P is protein: ##STR1##
Activity of thiol-dependent enzymes is a function of the availability of the thiols involved as expressed by the thiol/disulfide ratios of their thiol/disulfide redox buffers (upper arrow); interaction is complex, however, and activity is further dependent on additional factors such as substrate, ambient ions, and type of reducing thiol (membrane-bound enzymes, for example, are resistant to reduction by glutathione). Similarly, activity of disulfide-dependent enzymes (in parenthesis) is a function of the availability of disulfides, as expressed by the thiol/disulfide ratios of their redox pairs (lower arrow).
By the above mechanisms, endogenous thiol/disulfide redox buffers such as GSH/GSSG systems control the activity of many critical enzymes; thyroxine monodeiodinase is exemplary of thiol-dependent enzymes. Regulation of the activity of this enzyme by thiol/disulfide buffer controls the induction of a host of important enzymes, including HMG-CoA reductase, the branch-point enzyme for the isoprenoid pathway, which in turn regulates the production of essential isoprenoids such as steroid hormones, dolichol, cholesterol, and ubiquinone and the isoprenylation of proteins (6,7,8,9,10,11). Glycolysis is also controlled by thiol-dependent and disulfide-dependent enzyme systems; phosphofructokinase, for example, is inactivated by disulfides, whereas fructose-1,6-bis-phosphatase with the reverse enzyme activity is activated by certain disulfides. Thiol-dependent enzymes also directly and/or indirectly control isoprenoid and oligosaccharide biosynthesis and the synthesis and utilization of thyroid hormones.
One mechanism postulated to participate in the in vivo regulation of thiol/disulfide equilibria is the oxidation of thiol to disulfide catalyzed by microsomal flavin-containing mixed function monooxygenase (herein referred to as "monooxygenase"). This monooxygenase catalyzes, for example, the oxidation of cysteamine to its corresponding disulfide, cystamine. A comparable oxygenase activity thus appears to be critical to the regulation of at least some thiol- and disulfide-dependent enzymes in vivo.
Certain other thiols (glutathione or cysteine or N-acetyl-cysteine) have been demonstrated in vivo to inhibit neoplasia (Am. J. Med. 91(3C): 122S-130S, 1991); to inhibit replication of HIV in cell cultures (Proc. Natl. Acad. Sci. USA 87 (12): 4884-8, 1990); to be markedly elevated in preneoplastic/neoplastic hepatocytes (Mol. Carcin. 2 (3): 144-9, 1989); to influence the proliferation of human peripheral blood lymphocytes (HPBL) and T-cells (Am. J. Med. 91(3C): 140S-144S, 1991) to reverse inhibition of lymphocyte DNA synthesis by glutamate in cells from HIV-infected patients (Int. Immunol. 1(4): 367-72, 1989); to reduce infectivity of herpes virus in vitro (Acta. Virol. Praha. 11(6): 559-61, 1967); to suppress HIV expression in monocytes (Proc. Natl. Acad. Sci. 88: 986-990, 1991); and to be systemically deficient in HIV-infected individuals (Biol. Chem. Hoppe Seyler 370: 101-08, 1989 and The Lancet ii: 1294-97, 1989). Regulation of HMG-CoA reductase activity by thiols and disulfides is well-known; as noted above, thyroxine monodeiodinase is a thiol-dependent enzyme, and this enzyme controls the induction of HMG-CoA reductase (Eur. J. Biochem. 4: 273-278, 1968). Hypercholesteremia and atherosclerosis, leading factors in heart disease, are now clearly linked to HMG-CoA reductase activity, and treatment of these conditions with various regulators of HMG-CoA reductase is known. HMG-CoA reductase activity is also linked to neoplasia, most recently by evidence of its role in the transformation of cells by activation of Ras protein which regulates oncogene expression (Adv. Enzymol. 38: 373-412, 1973; Biochem. Soc. Trans. 17: 875-876, 1989; Science 245: 379-385, 1989; 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, Sabine ed. CRC Press, Inc., Boca Raton, Fla., U.S.A., pp. 245-257, 1983).